Project Summary Although largely asymptomatic, human cytomegalovirus (HCMV) can cause severe and even fatal disease in a subset of susceptible individuals. While great progress has been made in understanding essential stages of HCMV replication, a detailed description of many of these processes is lacking. Of particular interest in this proposal is cytoplasmic envelopment. To provide a molecular description of the events associated with cytoplasmic envelopment, it is important to identify the factors involved, both viral and cellular. Previous work has identified UL71 as an envelopment factor that potentially mediates membrane scission, as viruses lacking UL71 are trapped at various stages of budding. It is not known whether UL71 is sufficient itself for promoting scission or if it requires other viral and/or cellular factors. Many viruses utilize cellular machinery known as the endosomal sorting complexes required for transport (ESCRTs) as part of the envelopment process. While HCMV does not require the early ESCRT-I complex, it does require the late ESCRT Vps4. The role of the ESCRT-III complex, which is the major driver of membrane deformation and scission promoter, has not yet been investigated. Understanding the role, if any, for ESCRT-III during infection is essential for providing a detailed description of cytoplasmic envelopment. We hypothesize that UL71 recruits ESCRT-III and Vps4 to mediate scission of budding HCMV capsids. In support of this hypothesis, an interaction between UL71 and Vps4 has been reported. The experiments in this proposal will investigate if ESCRT-III subunits are required for HCMV replication, and if so at what stage. They will investigate the relationship, if any, between UL71 and ESCRT-III and define a function for the UL71-Vps4 interaction, as well as identify regions on UL71 important for function. These studies will further our understanding of the contribution of cellular factors to HCMV infection and potentially identify novel ways in which viral replication can be targeted. Taken together, these studies will lay the foundation for a mechanistic understanding of HCMV cytoplasmic envelopment.